The research outlined in all projects making up this program involves the use of aged rats, primary cultures of neurons or cardiomyocytes, and various cell lines to examine the role of oxidative stress in age-dependent alterations in brain, heart, and skeletal muscle. The projects are highly interactive, and sharing of tissues and cells in culture is common. In order to optimize the harvesting of tissues from aged animals, to prepare and maintain cell culture systems identically across projects, and to provide molecular biological techniques required in some projects, we plan to establish a new Core within the University's Tissue Culture/Hybridoma Facility. Aims of the core are: (1) to coordinate ordering of rats of various ages and optimize utilization of the tissues from these animals across the projects; (2) to prepare and maintain the primary neuronal and cardiac cell cultures and the PC 12, CHO, and HEK cells used across the projects; and (3) to assist in mRNA quantification in tissues and cells, to design and deliver antisense oligonucleotides to neurons in culture, and to infect neurons with viral constructs for the purpose of altering expression of specific proteins. Identification of biochemical and cellular events that are integral to the aging process clearly involves examination of tissues from animals undergoing "normal biological aging." Once specific age- related alterations have been observed, investigations into the mechanisms responsible for those alterations usually require model systems in which specific variables can be experimentally manipulated. Research strategies in several projects are designed to go back and forth between observations form in vivo aging and in vitro cell culture models that are amenable to mechanistic explorations. The role of oxidative stress in the aging process, particularly as it affects the regulation of Ca2+ in brain and muscle cells, unites all of the projects. Centralization of the procedures involved in obtaining both the in vivo aged tissues and the preparation of the cell culture models will both enhance efficiency and ensure that the cell culture models being tested within the projects Are prepared and maintained under identical conditions.